Abstract 6562: Identifying vulnerabilities to immune checkpoint inhibitors of oncogene-addicted non-small cell lung cancer subgroups

Abstract Purpose: Even though the treatment with PD-(L)1 axis blockers induces tumor response in approximately 20% of unselected lung cancer patients with advanced Non-Small Cell Lung Cancer (NSCLC), patients harbouring EGFR alterations or ALK-rearrangements have a poor response to immunotherapy. Tyrosine Kinase Inhibitors (TKIs) is the standard of care for these patients. However, resistance to TKIs is almost inevitable. The different clinical response to Immune Checkpoint Inhibitors (ICIs) in the different oncogene-addicted NSCLC subgroups may be explained by the composition and quality of the TME. Thus, we propose to investigate potential vulnerabilities and opportunities to overcome primary resistance to ICIs conferred by oncogene addiction. Methods: To identify mechanisms involved in the restricted immune response of these subgroups, we performed a sub-genome-scale in vivo CRISPR/Cas9 screening, using a lentiviral vector system that allowed selective CRISPR antigen removal (SCAR) from tumor cells after genome editing. Genetically engineered mouse models were used to isolate cell lines bearing Egfr exon 19 deletion, Egfr L860R missense mutation or the Eml4-Alk oncogene fusion. Modified cell lines with the CRISPR/Cas9 sgRNA library were implanted subcutaneously into the flank of immunodeficient NOD-scid IL2Rgnull and immunocompetent wild-type (WT) mice. A group of WT mice received ICI treatment at different days post-tumor challenge and sgRNA abundances between the different groups were compared. Results: As expected, both Egfr murine cell lines were sensitive to an EGFR-specific TKI, showing a decrease in phospho-Y1068-EGFR, demonstrating the oncogenic dependence on EGFR signaling for growth. However, when these cell lines were injected subcutaneously into immunocompetent mice, these tumors were resistant to PD1 blocking, mirroring the lack of response to human EGFR-mutant NSCLC to immunotherapy. To ensure an optimal coverage of the sgRNA library for in vivo studies, we estimated that around 55 mice for each experimental condition were necessary. Based on this estimation, an in vivo screen was performed and sgRNAs abundance are being analyzed to determine mechanisms of immune evasion in an Egfr oncogenic specific context. Cell lines bearing Eml4-Alk gene fusion were positive for TTF1 and SpC and negative for p63, confirming a lung adenocarcinoma phenotype. Phosphorylation of Stat3, Erk1/2 and Akt were also observed. The next steps include genetic modification of Eml4-Alk fusion cell lines with the CRISPR/Cas9 sgRNA library, followed by in vivo screening. Conclusion: We expect to identify new resistance mechanisms that could result in novel treatment strategies for these NSCLC subgroups. The identification of these targets will provide the opportunity to reprogram the TME and to improve the efficacy of immunotherapy in a subset of patients with lung adenocarcinoma. Citation Format: Inés Díaz-Cano, José Gracia, Patricia Cozar, Belén Revuelta, Nuria Carrizo, Laura García-Redondo, Joan Russo, Rita Manzano, Jose Garrido-Mesa, Daniel Meraviglia-Crivelli, Juan Dubrot, Luis Paz-Ares, Itziar Otano. Identifying vulnerabilities to immune checkpoint inhibitors of oncogene-addicted non-small cell lung cancer subgroups abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 6562.